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Biomedical subjects

C C Pollitt

Publications and source records attributed to C C Pollitt.

At least 19 recordsLinked to original sources
PubMed2007

Lamellar pro-inflammatory cytokine expression patterns in laminitis at the developmental stage and at the onset of lameness: innate vs. adaptive immune response.

REASONS FOR PERFORMING STUDY: Recent research has indicated that inflammation plays a role in the early stages of laminitis and that, similar to organ failure in human sepsis, early inflammatory mechanisms may lead to downstream events resulting in lamellar failure. Characterisation of the type of immune response (i.e. innate vs. adaptive) is essential in order to develop therapeutic strategies to counteract these deleterious events. OBJECTIVES: To quantitate gene expression of pro-inflammatory cytokines known to be important in the innate and adaptive immune response during the early stages of laminitis, using both the black walnut extract (BWE) and oligofructose (OF) models of laminitis. METHODS: Real-time qPCR was used to assess lamellar mRNA expression of interleukins-1beta, 2, 4, 6, 8, 10, 12 and 18, and tumour necrosis factor alpha and interferon gamma at the developmental stage and at the onset of lameness. RESULTS: Significantly increased lamellar mRNA expression of cytokines important in the innate immune response were present at the developmental stage of the BWE model, and at the onset of acute lameness in both the BWE model and OF model. Of the cytokines characteristic of the Th1 and Th2 arms of the adaptive immune response, a mixed response was noted at the onset of acute lameness in the BWE model, whereas the response was skewed towards a Th1 response at the onset of lameness in the OF model. CONCLUSIONS: Lamellar inflammation is characterised by strong innate immune response in the developmental stages of laminitis; and a mixture of innate and adaptive immune responses at the onset of lameness. POTENTIAL RELEVANCE: These results indicate that anti-inflammatory treatment of early stage laminitis (and the horse at risk of laminitis) should include not only therapeutic drugs that address prostanoid activity, but should also address the marked increases in lamellar cytokine expression.

Animals↗
PubMed2006

Changes in equine hindgut bacterial populations during oligofructose-induced laminitis.

In the horse, carbohydrate overload is thought to play an integral role in the onset of laminitis by drastically altering the profile of bacterial populations in the hindgut. The objectives of this study were to develop and validate microbial ecology methods to monitor changes in bacterial populations throughout the course of experimentally induced laminitis and to identify the predominant oligofructose-utilizing organisms. Laminitis was induced in five horses by administration of oligofructose. Faecal specimens were collected at 8 h intervals from 72 h before to 72 h after the administration of oligofructose. Hindgut microbiota able to utilize oligofructose were enumerated throughout the course of the experiment using habitat-simulating medium. Isolates were collected and representatives identified by 16S rRNA gene sequencing. The majority of these isolates collected belonged to the genus Streptococcus, 91% of which were identified as being most closely related to Streptococcus infantarius ssp. coli. Furthermore, S. infantarius ssp. coli was the predominant oligofructose-utilizing organism isolated before the onset of lameness. Fluorescence in situ hybridization probes developed to specifically target the isolated Streptococcus spp. demonstrated marked population increases between 8 and 16 h post oligofructose administration. This was followed by a rapid population decline which corresponded with a sharp decline in faecal pH and subsequently lameness at 24-32 h post oligofructose administration. This research suggests that streptococci within the Streptococcus bovis/equinus complex may be involved in the series of events which precede the onset of laminitis in the horse.

Animal Feed↗
PubMed2006

Equine laminitis induced with oligofructose.

REASONS FOR PERFORMING STUDY: Experimental induction of equine laminitis with a reliable and clinically relevant model should facilitate understanding of the disease. Successful induction with oligofructose (OF) could link pasture consumption to laminitis. OBJECTIVES: To determine whether alimentary administration of OF induces laminitis. METHODS: Twelve horses were dosed with OF and 6 received a sham (placebo) treatment. Clinical observations were made and blood collected at 4 h intervals over a 48 h study period. Stained sections of the hoof wall lamellae, examined by light microscopy, were graded for laminitis severity. RESULTS: All horses administered OF, but no sham-treated controls, developed clinical and histological laminitis. CONCLUSIONS AND POTENTIAL RELEVANCE: Alimentary overload with OF is a valid induction model for studying the pathogenesis of laminitis. A link is therefore established between field cases of laminitis and pasture fructan content.

Animals↗
PubMed2006

Magnetic resonance microscopy of the equine hoof wall: a study of resolution and potential.

REASONS FOR PERFORMING STUDY: Obtaining magnetic resonance images of the inner hoof wall tissue at the microscopic level would enable early accurate diagnosis of laminitis and therefore more effective therapy. OBJECTIVES: To optimise magnetic resonance imaging (MRI) parameters in order to obtain the highest possible resolution of the structures beneath the equine hoof wall. METHODS: Magnetic resonance microscopy (MRM) was performed in front feet from 6 cadaver horses using T2-weighted fast spin echo (FSE-T2), and T1-weighted gradient echo (GRE-T1) sequences. RESULTS: In T2 weighted FSE images most of the stratum medium showed no signal, however the coronary, terminal and sole papillae were visible. The stratum lamellatum was clearly visible and primary epidermal lamellae could be differentiated from dermal lamellae. CONCLUSION: Most structures beneath the hoof wall were differentiated. Conventional scanners for diagnostic MRI in horses are low or high field. However this study used ultra-high field scanners currently not available for clinical use. Signal-to-noise ratio (S/N) increases as a function of field strength. An increase of spatial resolution of the image results in a decreased S/N. S/N can also be improved with better coils and the resolution of high field MRI scanners will increase as technology develops and surface array coils become more readily available. POTENTIAL RELEVANCE: Although MR images with microscopic resolution were obtained ex vivo, this study demonstrates the potential for detection of lamellar pathology as it occurs. Early recognition of the development of laminitis to instigate effective therapy at an earlier stage and may improve the outcome for laminitic horses. Clinical MR is now readily available at 3 T, while 4 T, 7 T and 9 T systems are being used for human whole body applications.

Abattoirs↗
PubMed2005

Histopathology of oligofructose-induced acute laminitis in heifers.

Histopathology of the dermo-epidermal junction in the lamellar region of front claws was examined in 6 dairy heifers given an alimentary oligofructose overload and compared with sections from a control group of 6 heifers. Four of the 6 heifers administered oligofructose developed clinical signs of acute laminitis before they were euthanized. Postmortem samples from front claws were processed for histology. Eleven histopathologic characteristics were selected from the existing literature and used in a blinded evaluation of sections. In total, 104 front claw samples, including 8 samples from 2 cows having spontaneously occurring acute laminitis, were evaluated histologically using hematoxylin and eosin as well as periodic acid-Schiff staining. The major morphological features associated with oligofructose-induced acute clinical laminitis were stretching of lamellae, dermal edema, hemorrhage, changes in basal cell morphology, presence of white blood cells in dermis, and signs of basement membrane detachment. Changes at the lamellar junction of claw tissue affected by oligofructose-induced clinical laminitis resembled tissue from the 2 cows suffering from spontaneous acute clinical laminitis, and generally were consistent with existing descriptions of laminitis histopathology. Important exceptions to existing descriptions in the literature were stretching of lamellae and basement membrane changes. Not previously described, we considered these early signs of acute laminitis. In conclusion, this study documents that oligofructose-induced clinical laminitis is associated with histopathological changes at the lamellar interface. A weakened dermo-epidermal junction is a possible intermediate stage in the pathophysiology of bovine sole ulceration at the typical site.

Animals↗
PubMed2004

Equine laminitis: cryotherapy reduces the severity of the acute lesion.

REASONS FOR PERFORMING STUDY: The hypometabolic and vasoconstrictive effects of cryotherapy could prevent the development of laminitis. OBJECTIVES: To use distal limb cryotherapy to prevent laminitis induced by alimentary carbohydrate overload. METHODS: Laminitis was induced in 6 Standardbred horses that had one front limb continuously cooled in an ice/water mixture. Lameness evaluation, blinded lamellar histological grading and analysis for lamellar matrix metalloproteinase-2 (MMP-2) mRNA expression were used to evaluate the severity of laminitis. RESULTS: Cryotherapy was well tolerated and effective in cooling the feet. In each horse no lameness was observed in the treated limbs. Laminitis histology scores in the treated limbs were significantly less than those of the corresponding untreated forelimbs (P < 0.05). Laminitis histology scores in the treated limbs were also significantly less than those of the untreated limbs (fore- and hind) as a group (P < 0.05). Expression of MMP-2 mRNA in the iced feet was significantly (P < 0.05) less than that detected in the untreated feet. CONCLUSIONS: Cryotherapy, when applied to one foot, markedly reduced the severity of acute laminitis in this study. We propose that vasoconstriction (preventing delivery of haematogenous trigger factors) and hypometabolism (reduction in lamellar MMP activity) were the primary therapeutic mechanisms. POTENTIAL RELEVANCE: Although further research is needed, we suggest cryotherapy as a potentially effective prophylactic strategy in horses at risk of developing acute laminitis.

Animals↗
PubMed2004

Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study.

REASONS FOR PERFORMING STUDY: Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. OBJECTIVES: To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. METHODS: Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. RESULTS: Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. CONCLUSIONS: Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. POTENTIAL RELEVANCE: A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.

Animals↗
PubMed2004

Equine laminitis: cleavage of laminin 5 associated with basement membrane dysadhesion.

REASONS FOR PERFORMING STUDY: The key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. For this to happen the structural and adhesion proteins of the basement membrane zone must be altered. Which proteins and how damage to them leads to the lamellar separation of laminitis is unknown. OBJECTIVES: To investigate lamellar hemidesmosome and cytoskeleton damage and basement membrane dysadhesion using light microscopy (LM) and immunofluorescence microscopy (IFM). METHODS: Cryostat sections of lamellar tissues from 2 control and 6 Standardbred horses with oligofructose induced laminitis were studied using LM and IFM. Plectin, integrin alpha6 and BP230 antibody was used to label hemidesmosome intracellular plaque proteins and anti-BP180 and anti-laminin 5 (L5) was used to label anchoring filament (AF) proteins. Cytoskeleton intermediate filaments were labelled using anti-cytokeratin 14. The primary antibodies of selected sections were double labelled to show protein co-localisation. RESULTS: Laminitis caused reduction of transmembrane integrin alpha6, the AF proteins BP180 and L5, and failure of co-localisation of BP180 and L5. Proteins of the inner hemidesmosomal plaque, plectin and BP230, were unaffected. CONCLUSIONS: Loss of co-localisation of L5 and BP180 suggests that, during the acute phase of laminitis, L5 is cleaved and therefore, the AFs connecting the epidermis to the dermis, fail. Without a full complement of AFs separation at the lamellar dermo-epidermal junction occurs. POTENTIAL RELEVANCE: Suppressing or inhibiting metalloproteinase activity may prevent L5 cleavage and therefore the lamellar dermo-epidermal separation of laminitis.

Acute Disease↗
PubMed2004

Prolonged, continuous distal limb cryotherapy in the horse.

REASONS FOR PERFORMING STUDY: The recommended duration of cryotherapy in horses is currently extrapolated from human medicine. Prolonged, continuous cryotherapy (days rather than minutes) may be of therapeutic value if it is safe and well tolerated. OBJECTIVES: To evaluate the effect of prolonged, continuous application of ice and water to the equine distal limb. METHODS: A slurry of ice and water was applied to the right forelimb of 4 Standardbred horses for 48 h. Hoof temperature, ambient temperature and ice boot temperature were logged continuously and clinical observations recorded every 2 h. Lameness examinations were performed prior to application and 1 week, 6 months and 1 year after removal of the ice boot. RESULTS: Continuous cryotherapy was well tolerated and resulted in marked cooling of the treated foot. No significant variation in clinical parameters was noted, and no lameness or gross pathology noted in the treated limbs at any examination period. CONCLUSIONS: The continuous application of ice and water is a safe and effective means of cooling the equine distal limb. The extremely low, constant temperatures achieved may be of benefit in the management of various distal limb conditions. POTENTIAL RELEVANCE: Providing clinicians and research workers with a potent therapeutic/prophylactic modality for disorders of the equine distal limb.

Animals↗
PubMed2004

Equine laminitis: glucose deprivation and MMP activation induce dermo-epidermal separation in vitro.

REASONS FOR PERFORMING STUDY: Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis. OBJECTIVES: To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation. METHODS: Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subjected to tension and processed for transmission electron microscopy (TEM). RESULTS: Without glucose, or with APMA, explants under tension separated at the dermo-epidermal junction. This in vitro separation occurred via 2 different ultrastructural processes. Lack of glucose reduced hemidesmosomes (HDs) numbers until they disappeared and the basal cell cytoskeleton collapsed. Anchoring filaments (AFs), connecting the basal cell plasmalemma to the BM, were unaffected although they failed under tension. APMA activation of constituent lamellar MMPs did not affect HDs but caused AFs to disappear, also leading to dermo-epidermal separation under tension. CONCLUSIONS: Natural laminitis may occur in situations where glucose uptake by lamellar basal cells is compromised (e.g. equine Cushing's disease, obesity, hyperlipaemia, ischaemia and septicaemia) or when lamellar MMPs are activated (alimentary carbohydrate overload). POTENTIAL RELEVANCE: Therapies designed to facilitate peripheral glucose uptake and inhibit lamellar MMP activation may prevent or ameliorate laminitis.

Acute Disease↗
PubMed2004

Hoof wall wound repair.

REASONS FOR PERFORMING STUDY: Surgical stripping of the hoof wall results in a wound that heals remarkabley well. In contrast, lamellae recovering from laminitis are often deformed. Investigating lamellar wound healing may aid understanding of laminitis. OBJECTIVES: To document temporal changes in the lamellar basement membrane (BM), dermis and epidermis after surgery. METHODS: Wall strips were made in the dorsal hoof wall midline of 6 mature horses. Immunohistochemistry was used to document changes in the basement membrane (BM) and detect proliferation of epidermal cells in lamellar tissues harvested at intervals. A conforming metal plate was screwed to the hoof wall to maintain alignment of the wound edges. RESULTS: Wall stripping caused lamellar tips to snap and remain behind in the dermis along with the majority of the lamellar BM and some lamellar basal cells. Three days later the BM was intact and new lamellae had been reconstructed by proliferation of surviving epidermal cells. By 5 days the surface of the stripped zone was covered with yellow epidermis that subsequently thickened and hardened. Eventually the hoof wall deficit was replaced by new wall growing down from the coronet. The conforming metal plate and post operative analgesic ensured minimal lameness. CONCLUSIONS AND POTENTIAL RELEVANCE: In wall stripped lamellae the BM survives virtually intact and is used as a template for proliferating cells, from snapped-off lamellar tips, to migrate and quickly achieve repair to near normality. In laminitis epidermal dysadhesion and lamellar BM destruction occurs and lack of a functional BM template may explain the prolonged and abnormal repair of affected lamellae.

Acute Disease↗
PubMed2004

Epidermal cell proliferation in the equine hoof wall.

REASONS FOR PERFORMING STUDY: Current theories explaining how the hoof wall 'grows' and moves past the stationary distal phalanx are speculative and based on incomplete evidence. Movement in the lamellar region could occur by cell proliferation or an enzyme-based remodelling process. Since laminitis pathogenesis appears to involve increased transcription and activation of enzymes normally involved in tissue remodelling, it is important to know precisely which process dominates the lamellar region of the hoof.. OBJECTIVES: To investigate epidermal cell proliferation in the equine hoof wall and calculate a proliferative index (PI) for the coronet, lamellae and toe. METHODS: An analogue of thymidine, 5-bromo-2'-deoxyuridine (BRdU), was infused i.v. into 5 ponies. After tissue harvesting, BRdU (and therefore basal cell proliferation) was detected immunohistochemically using mouse anti-BRdU. PIs were calculated for the coronet and 10 levels of the dorsal hoof wall lamellae. RESULTS: The highest PIs (mean +/- s.e.) were in the coronet; 12.04% +/- 1.59 and proximal lamellae (7.13% +/- 1.92) and are therefore growth zones of the proximal hoof wall. PIs of more distal lamellae were 0.11% +/- 0.04 to 0.97% +/- 0.29; significantly lower (P = 0.05) than the lamellar growth zone. CONCLUSIONS: A 20-fold PI decrease between proximal and more distal lamellae suggests that the majority of the normal lamellae are nonproliferative and their main function is to suspend the distal phalanx within the hoof capsule. Remodelling within the hoof wall epidermal lamellae, which must occur as the hoof wall moves past the stationary distal phalanx, is a process not requiring epidermal cell proliferation. POTENTIAL RELEVANCE: A hoof lamellar epidermis that remodels using the same MMPs involved in laminitis pathogenesis implies that laminitis is a normal process out of control. Understanding MMP control and how the normal lamellar epidermis achieves this will help in the development of better laminitis preventative and treatment strategies.

Animals↗
PubMed2004

Equine laminitis: increased transcription of matrix metalloproteinase-2 (MMP-2) occurs during the developmental phase.

REASONS FOR PERFORMING STUDY: The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription. OBJECTIVES: To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue. METHODS: Total RNA was isolated, fragmented by RT-PCR, cloned into vector and sequenced. Rabbit anti-equine MMP-2 and labelled MMP-2 riboprobe were developed to analyse and quantitate MMP-2 expression. RESULTS: Western immunoblotting with anti-MMP-2 detected 72 kDa MMP-2 in hoof tissue homogenates and cross-reacted with human MMP-2. Immunohistochemistry and in situ hybridisation detected MMP-2 in the cytoplasm of basal and parabasal cells in close proximity to the lamellar basement membrane. Northern analysis and quantitative real-time PCR showed MMP-2 expression significantly (P < 0.01) elevated in laminitis affected tissues. CONCLUSION: The lamellar pathology of laminitis is associated with increased transcription of MMP-2. POTENTIAL RELEVANCE: Real-time PCR analysis of lamellar MMP-2 accurately monitors laminitis development at the molecular level and can be used diagnostically and for testing preventive strategies. Controlling increased MMP-2 transcription may ameliorate or prevent laminitis in high risk clinical situations. Our findings represent a warning to clinicians that the basement membrane lesion of laminitis is insidious and well under way before clinical signs are apparent.

Amino Acid Sequence↗
PubMed2004

Acute bovine laminitis: a new induction model using alimentary oligofructose overload.

Twelve dairy heifers were used to examine the clinical response of an alimentary oligofructose overload. Six animals were divided into 3 subgroups, and each was given a bolus dose of 13, 17, or 21 g/kg of oligofructose orally. The control group (n = 6) was sham-treated with tap water. Signs of lameness, cardiovascular function, and gastrointestinal function were monitored every 6 h during development of rumen acidosis. The heifers were euthanized 48 and 72 h after administration of oligofructose. All animals given oligofructose developed depression, anorexia, and diarrhea 9 to 39 h after receiving oligofructose. By 33 to 45 h after treatment, the feces returned to normal consistency and the heifers began eating again. Animals given oligofructose developed transient fever, severe metabolic acidosis, and moderate dehydration, which were alleviated by supportive therapy. Four of 6 animals given oligofructose displayed clinical signs of laminitis starting 39 to 45 h after receiving oligofructose and lasting until euthanasia. The lameness was obvious, but could easily be overlooked by the untrained eye, because the heifers continued to stand and walk, and did not interrupt their eating behavior. No positive pain reactions or lameness were seen in control animals. Based on these results, we conclude that an alimentary oligofructose overload is able to induce signs of acute laminitis in cattle. This model offers a new method, which can be used in further investigation of the pathogenesis and pathophysiology of bovine laminitis.

Acidosis↗
PubMed2002

Thermolysin activates equine lamellar hoof matrix metalloproteinases.

Cultured equine lamellar hoof explants secrete the pro-enzymes matrix metalloproteinase-2 (MMP-2, 72 kDa) and MMP-2 (92 kDa). Untreated explants remained intact when tested on a calibrated force transducer, but when treated with an MMP activator, developed "in-vitro laminitis", separating at the dermal-epidermal junction. Explants treated with the bacterial protease thermolysin separated dose-dependently; this was accompanied by activation of both MMP-2 and -9. Thermolysin-mediated MP activation did not occur in a cell-free system and was not inhibited by the addition of the MMP inhibitor and batimastat. These findings suggest that thermolysin-mediated gelatinase activation is not dependent on membrane-bound matrix metalloproteinase (MT-MMP) activation, providing further evidence that bacteria can produce potent MMP activators that probably facilitate host invasion.

Animals↗
PubMed2001

In vitro evidence for a bacterial pathogenesis of equine laminitis.

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation. These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis.

Animals↗
PubMed2001

In situ zymography: topographical considerations.

In situ gelatin zymography is a simple technique providing valuable information about the cellular and tissue localization of gelatinases. Until recently, the use of this technique has been confined to soft, relatively homogeneous tissue. In this report in situ zymography has been utilized to assess the sub-lamellar location of gelatinases in the hard, semi-keratinized epidermal layer and the adjacent soft connective tissue matrix of the dermis of the equine hoof. We show that alterations in the orientation at which the tissue is dipped and withdrawn from the emulsion cause profound alterations in emulsion thickness. Microscopic variations in the surface topography of frozen tissue sections also influence emulsion thickness making interpretation of the results difficult. Given these results, researchers must be aware of potential variations in zymographic analysis may be influenced by physical tissue parameters in addition to suspected gelatinase activity.

Animals↗